Abstract: Tenebrio molitor antifreeze protein (TmAFP) is considered a potential green kinetic inhibitor of natural gas hydrates due to its excellent ice recrystallization inhibition activity. However, the structure–activity relationship of TmAFP in inhibiting methane hydrate growth remains unclear. In this study, molecular dynamics simulations were performed to investigate the effects of mutating key threonine (THR) residues to tyrosine (TYR) in TmAFP on methane hydrate growth inhibition. In the THR array, residues at positions 27, 39, and 44 exhibit a greater influence on the thermal hysteresis activity of the antifreeze protein than other sites. Furthermore, the substitution with amino acids possessing larger side chains than THR, such as tyrosine, may interfere with the ice-binding process through steric hindrance. We constructed the complex systems of wild-type and T27Y, T39Y, and T44Y mutants with methane hydrate. The results show that wild-type TmAFP exhibits the strongest inhibitory effect on methane hydrate growth, while TYR point mutations significantly weaken this activity. Notably, although the T44Y mutant retains 80% of its thermal hysteresis activity (ice recrystallization inhibition ability), it almost completely loses its ability to inhibit methane hydrate growth, indicating no positive correlation between the thermal hysteresis activity of antifreeze protein and its methane hydrate inhibition activity. Further analysis reveals that the T44Y mutation, while preserving the structural compatibility of the ice-binding surface (IBS), significantly increases the overall conformational flexibility of the protein and shortens the lifetime of interfacial hydrogen bonds, thereby preventing stable adsorption onto the hydrate surface. In contrast, the T27Y and T39Y mutations primarily impair inhibition by disrupting the matching between key residue spacings on the IBS and the cage structures of methane hydrate. This study demonstrates that the inhibition of methane hydrate growth by TmAFP requires not only structural complementarity between the IBS and the hydrate surface, but also synergy between the overall rigidity of the protein and the anchoring ability of residues on the hydrate surface. These findings provide molecular-level theoretical guidance for the rational design of efficient and green antifreeze protein-based hydrate inhibitors.

Key words: Tenebrio molitor antifreeze protein, Tyrosine site-directed mutation, Methane hydrate, Molecular dynamics simulation, Inhibitory mechanism